This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cullin RING ligases (CRLs) comprise the largest subfamily of E3 ubiquitin ligases. In humans, six cullins (CUL1, 2, 3, 4A, 4B, and 5), two RBX-family RING proteins (RBX1 and 2), and hundreds of substrate receptors assemble into distinct CRLs that mediate ubiquitination of thousands of targets to regulate a vast array of cellular processes. CRL function is regulated by attachment of the ubiquitin-like protein (UBL) NEDD8 to a conserved Lys in a cullin's C-terminal domain. NEDD8 both enhances intrinsic CRL ubiquitination activity, and prevents CRL binding to the inhibitor CAND1. In humans, the NEDD8 cascade is known to contain a single E1 (NAE1-UBA3), and two E2s (Ubc12 and UBE2F). In yeast, only a single E2, Ubc12, has been found to work with the NEDD8 ortholog, Rub1. Despite the importance of CRL activation by NEDD8/Rub1, mechanisms underlying cullin ligation to NEDD8/Rub1 remain incompletely understood. A detailed mechanistic view is lacking, in part because two different proteins have been reported as being the E3 for NEDD8/Rub1 ligation to Cul1 or its yeast ortholog, Cdc53. One candidate E3 is Rbx1, which binds Cul1/Cdc53 and has a RING domain. However, Dcn1 was also identified as a Rub1 E3. The Dcn1 crystal structure revealed two domains, a UBA domain, and a "potentiating neddylation" (PONY) domain. The PONY domain alone was reported to bind Ubc12 and Cdc53, and is sufficient to enhance Cdc53~Rub1 levels in vivo and in lysates. Upon this discovery, it was suggested that Rbx1 may play a passive structural role in cullin modification by Rub1. However, Cdc53 can be modified in vitro without Dcn1, raising questions as to Dcn1's function as an E3. Thus, we are dissecting mechanisms underlying NEDD8/Rub1 ligation to Cul1/Cdc53.